A Role for TFIIIC Transcription Factor Complex in Genome Organization Ken-ichi Noma, Hugh P. Cam, Richard J. Maraia, Shiv I.S. Grewal  Cell  Volume 125, Issue 5, Pages 859-872 (June 2006) DOI: 10.1016/j.cell.2006.04.028 Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 1 Histone Modifications around the IR-L and IR-R Boundary Elements (A) A physical map of the mating-type region. The IR-L and IR-R inverted repeats surrounding the mat2/3 heterochromatic domain are shown as light blue arrows. The green box represents the cenH sequence that shares homology to centromeric repeats. Open boxes correspond to ORFs. (B and C) ChIP analysis showing the distribution of histone H3 and its modifications at IR-L and IR-R using antibodies against histone H3, H3 Lys9-acetyl (K9ac), H3 Lys14-acetyl (K14ac), H3K9-dimethyl (K9me), and H3K4-dimethyl (K4me). DNA isolated from ChIP and WCE fractions was subjected to multiplex PCR to amplify DNA fragments from the mating-type region (mat) and an act1 fragment serving as an amplification control. Numbered bars below the map indicate the primer pairs used in each PCR (Noma et al., 2001). The ratios of the mat and control act1 signals were used to calculate relative fold enrichment values plotted in alignment with the map of the IR elements. Cell 2006 125, 859-872DOI: (10.1016/j.cell.2006.04.028) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 2 TFIIIC Binding at the IR-R Element Is Required for Boundary Activity (A) Identification of boundary core sequence. Different portions of the IR-R were replaced with kan+ gene (green boxes; not drawn to scale). The B-boxΔ cells lack a 240-bp sequence that contains 5 B-boxes and do not carry the kan+ gene. Serial dilution plating assays in the presence and absence of FOA were done to measure the expression of ura4+ marker inserted just to the right of the IR-R. Growth on FOA plate indicates repression of the ura4+ gene. (B) B-boxes at the IR element. Red arrows indicate the positions of the B-boxes. The B-box sequences from the IR are aligned with the B-box consensus sequence present at tRNA genes (Hamada et al., 2001). Asterisks indicate conserved nucleotides. (C) Heterochromatin spreading beyond the IR lacking B-boxes. H3K9me and H3K4me levels as determined by ChIP are shown. (D) Localization of the TFIIIC component, Sfc6, at the IR. Levels of Sfc6 binding at IR-R were determined by ChIP. (E) B-boxes are required for Sfc6 binding at IR. Sfc6 levels at IR-R in wild-type (wt) or B-box-deficient strains (B-boxΔ) were measured by ChIP. Multiplex PCR was performed using primers that specifically amplify the IR-R element (primer pairs 73 for wild-type [Figure 2D] and BΔ for B-boxΔ strain [Figure 2C]) and act1 primers. Intensities of bands representing IR and act1 in ChIP and WCE lanes were used to calculate relative enrichment shown below ChIP lane. (F and G) Synthetic B-boxes confer boundary activity. Strain carrying IR-RΔ::3 X B-boxes was created similar to IR-Rs strain but containing three tandem copies of 11 nucleotides B-box consensus sequence (red triangles). Black bold line below each map indicates the PCR fragment used for ChIP analysis shown in Figure 2G. Cell 2006 125, 859-872DOI: (10.1016/j.cell.2006.04.028) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 3 Analysis of Transcripts Generated from the IR Element (A) Detection of transcripts from the IR. RT-PCR was performed using primer pairs 1–7. Arrowheads indicate B-box positions. Red wavy lines represent predicted transcripts based on results shown in Figures 3A and 3B. The asterisk indicates spliced transcript derived from the IR, as confirmed by DNA sequencing. PCR with genomic DNA as template is shown as a control (bottom). RT, reverse transcription. (B) Direction of IR transcription. Strand-specific RT-PCR was performed as described previously (Noma et al., 2004). Arrows indicate the direction of the primer used in first strand cDNA synthesis. The cenH primer pair was used as a positive control as both strands of cenH are known to be transcribed. (C) RNA Pol III subunit, Rpc11, does not localize to the IR-R, as determined by ChIP. (D) Localization of the RNA Pol II subunit, Rpb1, at the IR. Level of Rpb1 at the IR-R was determined by ChIP using the IR-(5) and mat 51 primer pairs. The mat 51 primer pair amplifies a sequence located at the silent mating-type region that is not transcribed even in the absence of heterochromatin. (E) Deletion of the B-box region results in reduced transcription at the IR. Transcript levels were analyzed by RT-PCR using RNA samples prepared from strains with (+) or without (−) the B-boxes at the IR-R. Experiments were performed using IR-LΔ swi6- strains. (F) Heterochromatin-independent transcription of the IR. The IR and cenH transcripts were analyzed by RT-PCR with RNA samples purified from wild-type (wt) or clr4Δ cells. Cell 2006 125, 859-872DOI: (10.1016/j.cell.2006.04.028) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 4 Genomewide Distributions of TFIIIC (Sfc6) and Pol III (Rpc130) (A) Chromosomal distribution profiles of Sfc6 and Rpc130. Schematic diagrams of S. pombe chromosomes are shown (top). Relative enrichments of Sfc6 and Rpc130 are plotted with respect to chromosome position. The centromere regions (cen1-cen3), the IR-R/L at the mating-type region, and TFIIIC-associated COC loci (see text) are indicated together with selective peaks corresponding to tRNA and 5S rRNA genes. (B) Distributions of Sfc6, Rpc130, H3K9me, and H3K4me at the mating-type region. The distributions of H3K9me and H3K4me were derived from high-resolution mapping by conventional ChIP analysis (Noma et al., 2001). (C) Distributions of Sfc6, Rpc130, H3K9me, and H3K4me at the subtelomere on the left arm of the chromosome 1. Green vertical lines and white boxes denote retrotransposon long terminal repeats (LTRs) and ORFs, respectively. SPAC212.11 (blue boxes), a recQ helicase, contains cenH-like sequence (red boxes). H3K9me and H3K4me distributions were described previously (Cam et al., 2005). (D) Distributions of Sfc6, Rpc130, H3K9me, and H3K4me at centromeres. Physical maps of S. pombe centromeres denoting centromeric otr regions comprised of dg and dh elements, imr and cnt, are shown (top), with the number of tRNA genes within each region shown directly below. Red vertical lines (tRNA genes); open boxes (ORFs); Inverted repeat elements flanking cen1 (IRC1-L/R) and cen3 (IRC3-L/R) (yellow arrows). Cell 2006 125, 859-872DOI: (10.1016/j.cell.2006.04.028) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 5 IRC1 Acts as Heterochromatin Boundary (A) Physical map of centromere 1. cen1 consists of the cnt1, imr1 repeats (imr1L and imr1R) and otr1 repeats (otr1L and otr1R). tRNA genes (red vertical lines); inverted repeat elements flanking cen1 (IRC1-L/R) (yellow arrows). (B) Mapping of TFIIIC (Sfc6) and histone modifications at IRC1-L. Schematic map of IRC1-L is shown (top panel). Numbered bars (c1–c8) below the map indicate primer pairs used in ChIP. (C) Identification of boundary sequence at the IRC1-L. A series of sequence deletion replaced with kan+ marker gene (light green boxes) was made at IRC1-L and adjoining regions. Serial dilution plating assays in the presence and absence of FOA were performed to measure expression of ura4+ marker inserted at the centromere-distal side of the tRNAphe gene. (D) Heterochromatin spreading in IRC1-LΔ cells. Levels of H3K9me and H3K4me in wild-type (wt) and cen1LΔ#3 strains determined by ChIP are shown in alignment with the map of IRC1-L region. Cell 2006 125, 859-872DOI: (10.1016/j.cell.2006.04.028) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 6 TFIIIC Associates with Multiple COC Loci Independent of Pol III (A) TFIIIC, but not Pol III, associates with COC loci. Localizations of TFIIIC components (Sfc6, Sfc1, and Sfc3) and Pol III components (Rpc11 and Rpc130) at the IR, tRNA genes, and COC loci were analyzed by conventional ChIP assays. ChIP and WCE signals were used to calculate enrichment of TFIIIC and Pol III. (B) Distributions of the Sfc6 and Rpc130 at and around the COC loci as determined by ChIP-chip analyses. ORFs (open boxes); putative B-boxes (red vertical lines). Annotations of genes containing Sfc6 binding peaks within their promoter regions are shown. Note that while COC loci are associated only with Sfc6, both Sfc6 and Rpc130 are localized at tRNAs, 5S rRNA, and srp7 (7SL signal recognition particle component), which reside near COC1 and COC2 loci. Cell 2006 125, 859-872DOI: (10.1016/j.cell.2006.04.028) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 7 A Role for TFIIIC in Genome Organization (A) TFIIIC bodies at the nuclear periphery. Deconvoluted immunofluorescent images of cells stained for either Flag-Sfc3 (red) or Sfc6-Myc (green) proteins were merged with DAPI signals (blue). (B) FISH analysis of COC loci in fission yeast nucleus. COC3, COC4, COC5, the mating-type region, and centromeres were visualized by FISH using Cy3-conjugated probes (red). FISH signals that lie at the edge of the DAPI signal were counted as loci associated with the nuclear periphery. More than 200 cells were counted for each probe. (C) Effect of COC3Δ on the localization of COC3 site within the nuclei of diploid cells. Deleted COC3 sequence is indicated in Figure 6B. Only cells displaying two discrete FISH signals as well as DAPI signal were investigated by microscopy. More than 100 cells were analyzed, and the results are summarized in graph (right panel). (D) A model for the role of TFIIIC in genome organization. The recruitment of TFIIIC to IRs and COC loci facilitates tethering these loci to the nuclear periphery. TFIIIC signal was also detected around the surface of the nucleolus. This nuclear peripheral tethering might restrict the spread of heterochromatin to surrounding sequences. TFIIIC localization at centromeric tRNA gene clusters could also contribute to barrier activity associated with these regions. TFIIIC localized at COC loci might facilitate the clustering of distant chromosomal loci into a few distinct nuclear bodies, thus creating a higher-order chromosome organization that imparts different properties on these regions including transcriptional regulation of nearby genes. Cell 2006 125, 859-872DOI: (10.1016/j.cell.2006.04.028) Copyright © 2006 Elsevier Inc. Terms and Conditions